The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of Fabs PGT 127 and 128 with Man9 at 1.65 and 1.29 Å resolution, respectively, and glycan binding data delineate a specific high mannose binding site. Fab PGT 128 complexed with a fully-glycosylated gp120 outer domain at 3.25 Å reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 IgGs may be mediated by cross-linking Env trimers on the viral surface.

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

Recent research from a variety of fields has revealed numerous biological roles for glycoconjugates. In order to investigate the functions of such molecules, extensive efforts have been directed toward the synthesis of natural and modified structures.2 To this end, the efficiency of oligosaccharide synthesis has been improved dramatically due to the development of new glycosylation reactions using a wide range of leaving groups and mild activating conditions. A trend utilizing the concept of chemoselective glycosylation has emerged.3 Such strategies take advantage of the differential chemical reactivities of glycosyl donors which can be controlled by protecting groups (ether-vs ester-type) and leaving groups.The most straightforward method among these is the direct use of glycosylation products as donors for the next coupling reaction, thereby negating the need for additional steps in the further manipulation of the anomeric center after each g l y~~~y l a t i o n .~~-~~~~~~J~~~~~~~-~~~ However, the length of the resulting sugar chain has been limited by the number of available leaving groups andor protecting groups.To overcome the limitation of existing strategies, we investigated the possibility of using two sets of chemically distinct (orthogonal) glycosyl donors and activation conditions (Scheme 1). The criteria for this concept to be practical are that (1) X should be unaffected under condition b required to activate the other donor (i.e., Y), and vice versa, and (2) both X and Y should remain compatible with subsequent manipulations of temporary protecting groups. For this orthogonal strategy, we (activalmr X) I A c , e o ycondition (activates ( b ) V)after nglycasylatiin cycles selected the phenylthio group for X and fluoride for Y as the leaving groups, and NIS-TfOH (or AgOTf)4,5 (condition a) and Cp2HfC12-AgC1046 (condition b) as promoters, respectively. For an initial attempt to demonstrate the feasibility of the strategy, N-phthaloyl (Phth) protected glucosamine (GlcN) derivatives were chosen as the monosaccharide units. This decision was based solely on the assumption that any stereochemical ambiguity could be eliminated by the strong 1,2-trans directing nature of the NPhth g r~u p .~~,~ However, it is to be stressed that the basic principle should be applicable to a wide variety of oligosaccharide structures. In addition, the biological significance of p-1,4 linked oligomers of glucosamine (e.g., chitin) is well recognized.* Also, the hydroxyl group at the C-4 position of GlcN is known to be relatively unreactive.2f Therefore, the construction of this type of oligosaccharide is a challenging task? The required GlcN derivatives 3-6 were synthesized according to the procedure described for closely related compounds.1°In order to assess the orthogonality of the above-mentioned combination of reactions, we examined glycosylations using 111J2 and 213J4 as donors. Thus, thioglycoside 1 was reacted (4) (a) Konradsson, P.; Mootoo, D. R.; McDevCt, R. E.; Fraser-Reid, B. J. Chem. SOC., Chem. Commun. 1990, 270-...

Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLuT4 seem to be internalized more slowly than non-sialylated GLuT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.